Invariant (i) natural killer (NK)T cells are a unique T lymphocyte subset that expresses an invariant T cell antigen receptor (TCR) alpha chain that is conserved between mice and humans. iNKT cells have been shown to respond to glycolipid antigens found on several types of bacteria, including LPS negative Sphingomonas spp., and antigens from the spirochete Borrelia burgdorferi (B. burgdorferi). iNKT cells have been also shown to be required for the clearance of Streptococcus pneumoniae. S. pneumoniae is a pathogen that is responsible for the death of approximately 3 million people worldwide each year. Over time, this pathogen has become resistant to many standard antibiotics, pointing to the importance of developing improved vaccines. Work from our laboratory has identified the glycolipids on S. pneumoniae to be the putative antigens recognized by the iNKT cell receptor, and synthetic versions of these antigens are being tested. The experiments in this proposal will further explore the iNKT cell response to S. pneumoniae, particularly the importance of these putative iNKT antigens in the clearance and survival from S. pneumoniae infection. Two adjacent genes in S. pneumoniae one known and the other predicted to encode the glycocsyltransferases that synthesize the mono- and diacyl glycerols recognized by iNKT cells, will be deleted. Infection by this mutant bacterial strain is predicted to abolish the antigen-dependent activation of iNKT cells, increasing its pathogenicity. Hypotheses: 1) iNKT cells are required for an optimal protective immune response against Strep, pneumoniae in mice. 2) The direct activation of iNKT cells by glycolipid antigen recognition is critical for a protective response, with indirect or cytokine-mediated iNKT cell activation playing only a minor role in clearance from infection. These hypotheses will be addressed through the following Specific Aims: Aim 1. Characterization of the iNKT cell response to S. pneumoniae infection. Aim 2. To identify the consequences of glycosyl transferase loss of function mutation on direct and indirect antigen mediated Valpha 14iNKT cell activation in vivo and in vitro.